Staphylococcus aureus is a common colonizer of the skin and nares of healthy individuals, but also a major cause of severe human infections. During interaction with the host, pathogenic bacteria must adapt to a variety of adverse conditions including nutrient deprivation. In particular, they encounter severe iron limitation in the mammalian host through iron sequestration by haptoglobin and iron-binding proteins, a phenomenon called “nutritional immunity.” In most bacteria, including S. aureus, the ferric uptake regulator (Fur) is the key regulator of iron homeostasis, which primarily acts as a transcriptional repressor of genes encoding iron acquisition systems. Moreover, Fur can control the expression of trans-acting small regulatory RNAs that play an important role in the cellular iron-sparing response involving major changes in cellular metabolism under iron-limiting conditions. In S. aureus, the sRNA IsrR is controlled by Fur, and most of its predicted targets are iron-containing proteins and other proteins related to iron metabolism and iron-dependent pathways. To characterize the IsrR targetome on a genome-wide scale, we combined proteomics-based identification of potential IsrR targets using S. aureus strains either lacking or constitutively expressing IsrR with an in silico target prediction approach, thereby suggesting 21 IsrR targets, of which 19 were negatively affected by IsrR based on the observed protein patterns. These included several Fe-S cluster- and heme-containing proteins, such as TCA cycle enzymes and catalase encoded by katA. IsrR affects multiple metabolic pathways connected to the TCA cycle as well as the oxidative stress response of S. aureus and links the iron limitation response to metabolic remodeling. In contrast to the majority of target mRNAs, the IsrR-katA mRNA interaction is predicted upstream of the ribosome binding site, and further experiments including mRNA half-life measurements demonstrated that IsrR, in addition to inhibiting translation initiation, can downregulate target protein levels by affecting mRNA stability.

Seemingly simple bacteria mount intricate adaptive responses when exposed to physical stress or nutrient limitation, and the activation of these responses is governed by complex signal transduction networks. Upon entry into the stationary growth phase, the soil bacterium Bacillus subtilis may develop natural competence, form biofilms or stress-resistant cells, or ultimately trigger a cellular differentiation program leading to spore formation. Master regulators, such as Spo0A, ComK, SinR, and SigB, constantly monitor the bacterium's environment and then determine appropriate adaptive responses. Here, we show that exposure of B. subtilis to visible light and other stresses triggers a general stress response-dependent block in competence development. SigB serves as an "emergency system" to silence inappropriate expression of an alternative developmental program in the face of unfavorable conditions. In particular, we document a stress-dependent molecular mechanism that prevents accumulation of the central competence regulator ComK via expression of a SigB-driven antisense RNA (as-comK, S365) which is part of a noncontiguous operon. IMPORTANCE:Bacillus subtilis exhibits a large number of different specific and general adaptation reactions, which need to be well balanced to sustain survival under largely unfavorable conditions. Under specific conditions, natural competence develops, which enables B. subtilis to actively take up exogenous DNA to integrate it into its own genome. In contrast to this specific adaptation, the general stress response is induced by a variety of exogenous stress and starvation stimuli, providing comprehensive protection and enabling survival of vegetative B. subtilis cells. In the present work, we reveal the molecular basis for the interconnection of these two important responses in the regulatory network. We describe that the master regulator of the general stress response SigB is activated by physiological stress stimuli, including daylight and ethanol stress, leading to the inactivation of the competence master regulator ComK by transcriptional anti-sense regulation, showing a strict hierarchy of adaptational responses under severe stress.

Diseases can be caused by molecular perturbations that induce specific changes in regulatory interactions and their coordinated expression, also referred to as network rewiring. However, the detection of complex changes in regulatory connections remains a challenging task and would benefit from the development of novel nonparametric approaches. We develop a new ensemble method called BoostDiff (boosted differential regression trees) to infer a differential network discriminating between two conditions. BoostDiff builds an adaptively boosted (AdaBoost) ensemble of differential trees with respect to a target condition. To build the differential trees, we propose differential variance improvement as a novel splitting criterion. Variable importance measures derived from the resulting models are used to reflect changes in gene expression predictability and to build the output differential networks. BoostDiff outperforms existing differential network methods on simulated data evaluated in four different complexity settings. We then demonstrate the power of our approach when applied to real transcriptomics data in COVID-19, Crohn's disease, breast cancer, prostate adenocarcinoma, and stress response in Bacillus subtilis. BoostDiff identifies context-specific networks that are enriched with genes of known disease-relevant pathways and complements standard differential expression analyses. Availability and implementation BoostDiff is available at https://github.com/scibiome/boostdiff_inference.

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a “small colony variant” phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.

ABSTRACT Bacillus subtilis is a Gram-positive bacterium that is well-known for its high secretory capacity. Although secretion of homologous proteins is extremely efficient, secretion of heterologous proteins imposes what is known as “secretion stress” on the cells. This is not desirable in an industrial setting, because the bacterial secretion stress responses can adversely affect product yields. So far, the main approach to reduce such counterproductive secretion stress responses has been to remove components of the CssR-CssS two-component regulatory system, including the CssR-CssS-regulated quality control proteases HtrA and/or HtrB. However, there are several examples indicating that a complete loss of these proteases does not correlate with improved protein secretion or a reduced secretion stress response. In this study, an alternative approach to limit the effects of secretion stress by modulating the protease activity of HtrA rather than completely removing this protease was investigated. Indeed, expression of the proteolytically inactive HtrA led to a generally improved fitness of the bacteria when producing the heterologous α-amylase AmyQ. Moreover, compared to control strains, the strain expressing the mutant HtrA provided higher AmyQ yields, which correlated with superior α-amylase enzymatic activity in shake flasks and fermenter-scale cultivations. Proteome analysis of the engineered strains revealed critical differences in stress responses and metabolism. Altogether, our present findings show how proteolytically inactive HtrA can be employed to achieve improved bacterial fitness and higher enzyme yields. IMPORTANCE In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.

Streptococcus pneumoniae two-component regulatory systems (TCSs) are important systems that perceive and respond to various host environmental stimuli. In this study, we have explored the role of TCS09 on gene expression and phenotypic alterations in S. pneumoniae D39. Our comparative transcriptomic analyses identified 67 differently expressed genes in total. Among those, agaR and the aga operon involved in galactose metabolism showed the highest changes. Intriguingly, the encapsulated and nonencapsulated hk09-mutants showed significant growth defects under nutrient-defined conditions, in particular with galactose as a carbon source. Phenotypic analyses revealed alterations in the morphology of the nonencapsulated hk09- and tcs09-mutants, whereas the encapsulated hk09- and tcs09-mutants produced higher amounts of capsule. Interestingly, the encapsulated D39∆hk09 showed only the opaque colony morphology, while the D39∆rr09- and D39∆tcs09-mutants had a higher proportion of transparent variants. The phenotypic variations of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 are in accordance with their higher numbers of outer membrane vesicles, higher sensitivity against Triton X-100 induced autolysis, and lower resistance against oxidative stress. In conclusion, these results indicate the importance of TCS09 for pneumococcal metabolic fitness and resistance against oxidative stress by regulating the carbohydrate metabolism and thereby, most likely indirectly, the cell wall integrity and amount of capsular polysaccharide.

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology.

Many bacteria encode so-called cold shock proteins. These proteins are characterized by a conserved protein domain. Often, the bacteria have multiple cold shock proteins that are expressed either constitutively or at low temperatures. In the Gram-positive model bacterium Bacillussubtilis, two of three cold shock proteins, CspB and CspD, belong to the most abundant proteins suggesting a very important function. To get insights into the role of these highly abundant proteins, we analyzed the phenotypes of single and double mutants, tested the expression of the csp genes and the impact of CspB and CspD on global gene expression in B. subtilis. We demonstrate that the simultaneous loss of both CspB and CspD results in a severe growth defect, in the loss of genetic competence, and the appearance of suppressor mutations. Overexpression of the third cold shock protein CspC could compensate for the loss of CspB and CspD. The transcriptome analysis revealed that the lack of CspB and CspD affects the expression of about 20% of all genes. In several cases, the lack of the cold shock proteins results in an increased read-through at transcription terminators suggesting that CspB and CspD might be involved in the control of transcription termination.

Streptococcus suis is an important pathogen of pigs that, as a zoonotic agent, can also cause severe disease in humans, including meningitis, endocarditis, and septicemia. We report complete and annotated genomes of S. suis strains 10, 13-00283-02, and 16085/3b, which represent the highly prevalent serotypes cps2, cps7, and cps9, respectively.

SUMMARY Genoscapist is a web-tool generating high-quality images for interactive visualization of hundreds of quantitative profiles along a reference genome together with various annotations. Relevance is demonstrated by deployment of two websites dedicated to large condition-dependent transcriptome datasets available for Bacillus subtilis and Staphylococcus aureus. AVAILABILITY Websites and source code freely accessible at https://genoscapist.migale.inrae.fr.

SummaryUpon competence‐inducing nutrient‐limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence‐assisted cell sorting (FACS). Using RNA‐seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post‐transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK‐binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA‐seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.

Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

The Gram-positive bacterium Bacillus subtilis is frequently exposed to hyperosmotic conditions. In addition to the induction of genes involved in the accumulation of compatible solutes, high salinity exerts widespread effects on B. subtilis physiology, including changes in cell wall metabolism, induction of an iron limitation response, reduced motility and suppression of sporulation. We performed a combined whole-transcriptome and proteome analysis of B. subtilis 168 cells continuously cultivated at low or high (1.2 M NaCl) salinity. Our study revealed significant changes in the expression of more than one-fourth of the protein-coding genes and of numerous non-coding RNAs. New aspects in understanding the impact of high salinity on B. subtilis include a sustained low-level induction of the SigB-dependent general stress response and strong repression of biofilm formation under high-salinity conditions. The accumulation of compatible solutes such as glycine betaine aids the cells to cope with water stress by maintaining physiologically adequate levels of turgor and also affects multiple cellular processes through interactions with cellular components. Therefore, we additionally analysed the global effects of glycine betaine on the transcriptome and proteome of B. subtilis and revealed that it influences gene expression not only under high-salinity, but also under standard growth conditions.

Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent.

Garlic plants (Allium sativum L.) produce antimicrobial compounds, such as diallyl thiosulfinate (allicin) and diallyl polysulfanes. Here, we investigated the transcriptome and protein S-thioallylomes under allicin and diallyl tetrasulfane (DAS4) exposure in the Gram-positive bacterium Bacillus subtilis. Allicin and DAS4 caused a similar thiol-specific oxidative stress response, protein and DNA damage as revealed by the induction of the OhrR, PerR, Spx, YodB, CatR, HypR, AdhR, HxlR, LexA, CymR, CtsR, and HrcA regulons in the transcriptome. At the proteome level, we identified, in total, 108 S-thioallylated proteins under allicin and/or DAS4 stress. The S-thioallylome includes enzymes involved in the biosynthesis of surfactin (SrfAA, SrfAB), amino acids (SerA, MetE, YxjG, YitJ, CysJ, GlnA, YwaA), nucleotides (PurB, PurC, PyrAB, GuaB), translation factors (EF-Tu, EF-Ts, EF-G), antioxidant enzymes (AhpC, MsrB), as well as redox-sensitive MarR/OhrR and DUF24-family regulators (OhrR, HypR, YodB, CatR). Growth phenotype analysis revealed that the low molecular weight thiol bacillithiol, as well as the OhrR, Spx, and HypR regulons, confer protection against allicin and DAS4 stress. Altogether, we show here that allicin and DAS4 cause a strong oxidative, disulfide and sulfur stress response in the transcriptome and widespread S-thioallylation of redox-sensitive proteins in B. subtilis. The results further reveal that allicin and polysulfanes have similar modes of actions and thiol-reactivities and modify a similar set of redox-sensitive proteins by S-thioallylation.

The major human pathogen Staphylococcus aureus is a widespread commensal bacterium but also the most common cause of nosocomial infections. It adapts to the different host niches through a complex gene regulatory network. We show here that the Rho transcription termination factor, which represses pervasive antisense transcription in various bacteria, including S. aureus , plays a role in controlling SaeRS-dependent virulence gene expression. A Rho-deficient strain produces larger amounts of secreted virulence factors in vitro and shows increased virulence in mice. We also show that treatment of S. aureus with the antibiotic bicyclomycin, which inhibits Rho activity and is effective against Gram-negative bacteria, induces the same changes in the proteome as observed in the Rho-deficient strain. Our results reveal for the first time a link between transcription termination and virulence regulation in S. aureus , which implies a novel mechanism by which an antibiotic can modulate the expression of virulence factors.